Heaves, an asthma-like equine disease, involves
airway smooth muscle remodeling
Bérénice Herszberg, DMV,* David Ramos-Barbón, MD, PhD,* Meiyo Tamaoka, MD,
James G. Martin, MD, and Jean-Pierre Lavoie, DMV Montreal, Quebec, Canada
Mechanisms of asthma and
allergic inflammation
Background: Increased airway smooth muscle mass is a
prominent feature of asthmatic airway remodeling. Airway
smooth muscle hyperplasia occurs in rodent models of
experimental asthma, but the relevance of such finding to
spontaneously occurring disease in large mammals is unknown.
Objective: We examined horses with heaves, a naturally
occurring equine asthma related to sensitization and exposure
to moldy hay. We hypothesized that airway remodeling occurs
in heaves and shares disease mechanisms with asthma.
Methods: We quantified the airway smooth muscle mass and
the numbers of proliferating and apoptotic airway smooth
muscle cells in 5 horses with heaves and 5 control horses using
morphometric techniques. Cell proliferation was detected in
tissue sections by immunostaining for proliferating cell nuclear
antigen, and apoptotic cells were detected by terminal
deoxynucleotidyl transferase-mediated dUTP nick end labeling
of fragmented DNA. Both signals were colocalized with smooth
muscle specific a-actin.
Results: Horses with heaves had a significant increase in the
amount of smooth muscle in the airways (nearly triple that of
the controls) associated with increased myocyte proliferation
(7-fold proliferating cell nuclear antigen–positive airway
myocytes) and apoptosis (6-fold).
Conclusion: Heaves involves airway smooth muscle growth
associated with myocyte hyperplasia, which may contribute to
the growth, and increased myocyte apoptosis that may reflect
a compensatory mechanism serving to limit the abnormal
smooth muscle growth.
Clinical implications: Airway smooth muscle remodeling in
heaves may be involved in the mechanism of airway
hyperresponsiveness and chronic lung function impairment in a
From the Meakins-Christie Laboratories, McGill University; and Faculté de
Médecine Vétérinaire, Université de Montréal, St-Hyacinthe.
*These authors contributed equally to this work.
Supported by the Richard and Edith Strauss Canada Foundation and the
Canadian Institutes of Health Research. Dr Ramos-Barbón is the recipient
of a Canadian Lung Association/BI/Pfizer fellowship and is currently
supported by an investigator’s contract of the National Health System of
Spain (Fondo de Investigaciones Sanitarias, Fund #CP04/00313).
Disclosure of potential conflict of interest: J. G. Martin has received grant
support from the Canadian Institutes of Health Research and the Canadian
Cystic Fibrosis Foundation. M. Tamaoka has received grant support from
the Canadian Institutes of Health. J. P. Lavoie has received grant support
from the Natural Sciences and Engineering Research Council. The rest of
the authors have declared that they have no conflict of interest.
Received for publication June 25, 2005; revised March 28, 2006; accepted for
publication March 31, 2006.
Available online May 28, 2006.
Reprint requests: James G. Martin, MD, Meakins-Christie Laboratories,
McGill University, 3626 Saint Urbain, Montreal, QC, H2X 2P2, Canada.
E-mail: [email protected].
0091-6749/$32.00
Ó 2006 American Academy of Allergy, Asthma and Immunology
doi:10.1016/j.jaci.2006.03.044
382
way comparable to human asthma. (J Allergy Clin Immunol
2006;118:382-8.)
Key words: Airway obstruction, apoptosis, animal disease models,
hyperplasia, smooth muscle cells
Asthma is a chronic inflammatory condition associated
with airway remodeling,1 a complex series of structural
changes in the airways, which includes increases in the
amount of airway smooth muscle (ASM) corrected by airway size (ASM mass). The increase in ASM mass is likely
important in the causation of airways hyperresponsiveness, as suggested by mathematical modeling of airway
mechanics2-4 and experimental data demonstrating a correlation between smooth muscle growth and airway hyperresponsiveness.5 Data from the Brown Norway (BN)
rat model of experimental asthma also suggest that the increase in ASM mass is caused at least in part by myocyte
hyperplasia.6
Although rat and mouse models have contributed
valuable data on biological mechanisms of ASM growth
that may operate in human asthma, the relevance of rodentbased experimental models to human airway remodeling may be influenced by factors related to animal size
and cell turnover. Rodent models of induced disease may
also have limitations in representing the pathogenesis of
actual asthma as a naturally occurring, chronic disease.
Here we hypothesized that heaves, a spontaneously occurring equine asthma-like disease, involves ASM remodeling. Heaves is an obstructive pulmonary disease that
develops in horses exposed to moldy hay, characterized
by chronic airway inflammation, airway hyperresponsiveness, and episodes of bronchoconstriction that may
improve in response to b2-adrenergic agonists and corticosteroids.7-9 The respiratory symptoms are associated with
elevated levels of IgE in bronchoalveolar lavage10,11 and
serum,12 supporting the role of type I hypersensitivity reactions to inhaled molds and fungi in dusty hay. Early
removal from hay exposure may be accompanied by remission of symptoms, whereas continued exposure may
lead to irreversible airflow limitation. Thus, heaves, similarly to asthma in human beings, may involve remodeling
of the airway wall as part of its pathogenesis. Horses with
heaves may therefore be a suitable animal model of asthmatic airway remodeling, having the characteristics of a
chronic and spontaneously occurring disease. In this study,
we analyzed the smooth muscle mass and the frequency of
myocyte proliferation and apoptosis in airway specimens
obtained from horses with heaves. We found a significantly increased ASM mass compared with controls, as
Herszberg et al 383
J ALLERGY CLIN IMMUNOL
VOLUME 118, NUMBER 2
well as evidence of hyperplasia and increased apoptosis of
ASM cells.
METHODS
Lung specimens
Lung tissue blocks sampling airways of different sizes were
harvested at autopsy from 5 horses with a diagnosis of heaves based
on a history of chronic respiratory disease, compatible clinical
manifestations, and abnormal lung function. These horses were
kept in pasture during the summer months and were stabled during
the winter, and had an increase in neutrophils (>5%) in bronchoalveolar lavage fluid when stabled and fed hay. Lung tissues from 5
adult control horses were obtained from a local slaughterhouse. The
control horses had no clinical evidence of respiratory disease, and
the absence of obvious respiratory illness was confirmed by gross
pathology. Both heaves-affected and control horses were older than
10 years (range, 12-21 years) with the exception of 1 control horse
(3 years), and both groups were made up of male and female horses.
The tissue specimens were formalin-fixed and paraffin-embedded.
Detection of ASM cell proliferation and
apoptosis, and signal colocalization
Immunohistochemistry was performed for the colocalization of
proliferating cell–associated nuclear antigen (PCNA) with smooth
muscle a-actin (a-SMA). The following mAbs were used: clone 1A4
to a-SMA (Sigma-Aldrich, St Louis, Mo) and clone Ab-1 to PCNA
(Calbiochem, San Diego, Calif). The tissue sections underwent a
high-temperature epitope unmasking treatment in antigen retrieval
buffer (Vector Laboratories, Burlingame, Calif) at 95°C for 30
minutes. Cell membranes were permeabilized in 0.2% Triton X-100
(Sigma-Aldrich) in pH 7.6 Trizma buffer. The tissue sections were
blocked in universal blocking solution (Dako, Carpinteria, Calif)
supplemented with 20% normal goat serum (Vector Laboratories)
and incubated with anti-PCNA antibody at 37°C for 1 hour. On
negative control sections, the primary antibody was replaced by a
mouse isotype IgG control at the same concentration. The detection
system consisted of a biotinylated goat antimouse IgG polyclonal
antibody (BD Biosciences, Mississauga, Québec, Canada), an avidin/
biotin-alkaline phosphatase complex (Vector Laboratories), and a
BCIP/NTB chromogen substrate (Vector Laboratories), which produces a violet precipitate. The PCNA immunostaining was followed
by immunostaining for a-SMA using the same avidin/biotin-alkaline
phosphatase complex detection method with Vector-Red chromogen
substrate (Vector Laboratories) as a developer. The sections were
then counterstained with methyl green, dehydrated, and mounted.
Apoptotic cells were detected by terminal deoxynucleotidyl
transferase-mediated dUTP nick end labeling of fragmented DNA
(TUNEL; Apop-Tag kit; Qbiogene Inc, Carlsbad, Calif) as per the
manufacturer’s instructions, and the signal was developed with
diaminobenzidine-nickel (Vector Laboratories). Colocalization with
a-SMA followed as for PCNA.
Morphometry was performed on cross-sections of complete
bronchioles and small to medium bronchi, and arc sectors of larger
bronchi. Each airway was mapped with partially overlapping microscopic fields recorded as calibrated digital image files, and the
complete airway was then reconstructed as a single large image using
commercial software (Adobe Photoshop; Adobe Systems Inc, San
José, Calif). a-Actin immunostained ASM bundles were subtracted
from the rest of the tissue components, and the total ASM surface
area and length of the airway basement membrane were measured
by using Image-Pro Plus analysis software (MediaCybernetics,
Carlsbad, Calif). To standardize for airway size, the ASM surface
area and the counts of PCNA1 and TUNEL1 cells were divided by
the basement membrane perimeter squared (PBM2). The basement
membrane perimeter is a constant dimension in an airway section,
regardless of the degree of ASM contraction and lung inflation.13
For arc sectors of airways larger than the tissue blocks, an estimate
of airway size was geometrically determined by fitting the airway
arc sector into an ellipse and applying the following formula:
PBM 5
ARCBM E
ARCE
where PBM is the estimated basement membrane perimeter of the
complete airway section, ARCBM is the measured length of the irregularly shaped basement membrane contained in the airway sample arc
sector, ARCE is the length of the perfect elliptic arc that fits the airway
arc, and E is the perimeter of the complete ellipse.
Statistical analysis
Data are expressed as means 6 SEMs. PCNA1 and TUNEL1 cell
counts are expressed as cells/mm2. ASM mass is a dimensionless
index measured as ASM surface area divided by PBM2. Measurements
were performed in 6.4 6 0.7 airways/horse (range, 4-10) made up of
different airway sizes. Group comparisons were analyzed with the Student t test. Associations were analyzed by least-square linear regression
or best fit nonlinear regression and the coefficient of correlation (R) or
the coefficient of determination (R2). P values less than .05 were considered significant. Data analysis was performed with SPSS (SPSS Inc,
Chicago, Ill) and SigmaPlot (Systat Software Inc, Richmond, Calif).
RESULTS
ASM mass
The lung sections of horses with heaves showed
evidence of increased mucus production, epithelial damage,
plugging of the airway lumen with mucous material and
epithelial debris, presence of inflammatory cell infiltrates
in the airway wall and in a peribronchial location, and an
overall airway wall thickening affecting all tissue layers
(Fig 1, A and B). To demonstrate airway remodeling, we
quantitatively compared the ASM mass. The horses with
heaves had a 2.8-fold increase compared with the control
horses (9.15 6 1.38 1023 vs 3.21 6 0.23 1023, respectively; P 5 .003; Fig 1, C).
ASM cell proliferation and apoptosis
To test the hypothesis that myocyte hyperplasia may
contribute to ASM growth in heaves, we colocalized
PCNA immunodetection with a-SMA (Fig 2, A and B)
and measured the numbers of PCNA1 smooth muscle
cells corrected by PBM2. The PCNA1 cells/mm2 were
Mechanisms of asthma and
allergic inflammation
Quantitative morphology
Abbreviations used
a-SMA: Smooth muscle a-actin
ASM: Airway smooth muscle
BN: Brown Norway
PCNA: Proliferating cell–associated nuclear antigen
TUNEL: Terminal deoxynucleotidyl transferase-mediated
dUTP nick end labeling
384 Herszberg et al
J ALLERGY CLIN IMMUNOL
AUGUST 2006
Mechanisms of asthma and
allergic inflammation
FIG 1. ASM growth in heaves. A, Small bronchus of a horse with
heaves. B, Size-matched bronchus in a control horse. C, ASM
mass, heaves vs control groups. Scale bars: 250 mm. *P < .05.
increased in heaves by 7.2-fold compared with the control
horses (6.41 6 1.26 vs 0.89 6 0.27 cells/mm2, respectively; P 5 .003; Fig 2, C).
Because apoptosis may participate in smooth muscle
cell turnover and the maintenance of tissue homeostasis,
FIG 2. Immunostaining for PCNA (nuclear dark signal) colocalized
with a-SMA (cytoplasmic red signal). A, High-magnification detail.
B, Equivalent microscopic field in a size-matched airway of a
control horse. C, PCNA1 cell numbers/mm2. Scale bars: 50 mm.
*P < .05.
we tested the hypothesis that an alteration in the rate
of myocyte apoptosis may participate in the abnormal
growth of ASM detected in heaves. Using an approach
similar to PCNA, the TUNEL1 nuclear signal of apoptotic
Herszberg et al 385
Mechanisms of asthma and
allergic inflammation
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VOLUME 118, NUMBER 2
FIG 3. Apoptosis detected by TUNEL and colocalized with a-SMA.
The plot represents TUNEL1 cells/mm2 in heaves vs controls.
*P < .05.
cells was colocalized with a-SMA, and the frequency of
apoptotic ASM cells was measured. There was a 6-fold
increase in the TUNEL1 cells/mm2 in the horses with
heaves compared with the controls (2.34 6 0.90 vs 0.39
6 0.12; P 5 .0004; Fig 3).
Effect of airway size on remodeling changes
In the control horses, the ASM mass and the normalized
numbers of PCNA1 and TUNEL1 cells/mm2 were
approximately constant at the different airway sizes
analyzed, resulting in an almost flat linear regression
function (slope 5 20.04, 20.05, and 20.02 for the ASM
mass, PCNA1, and TUNEL1 cells/mm2, respectively;
Fig 4). This is consistent with using PBM2 for data normalization as an appropriate standardization for airway size.
Consistently with the differences observed between
the heaves and control groups, the respective regression
functions that relate ASM mass and the numbers of
proliferating and apoptotic cells with airway size were
displaced upward to higher ordinate values in the heaves
group (Fig 4). In the presence of heaves, the airway size
influenced the increases in ASM mass (R 5 20.68; P 5
.001) and PCNA1 cells/mm2 (R 5 20.45; P 5 .002);
the increments were larger the smaller the airways.
These data suggest that heaves involves hyperplastic
smooth muscle growth in all airway sizes, but the effect
is more marked in small airways. It is unclear whether
this is also the case for the increase in TUNEL1 cells/
mm2, because the data are inconclusive about the influence of airway size (P 5 .656; power 5 14.9%; Fig 4, C).
Relationship between proliferation
and apoptosis
The ASM mass correlated with the numbers of PCNA1
cells/mm2 (R 5 0.57; P < .0001; Fig 5, A), supporting the
idea that myocyte hyperplasia contributes to the growth of
ASM. A weak positive association was found between the
numbers of proliferating and apoptotic smooth muscle
cells (R 5 0.34; P 5 .042; Fig 5, B). This result was
inferred by correlating the numbers of PCNA1 and
TUNEL1 cells/mm2 in airways matched by size (<10%
FIG 4. Regression analysis of the ASM mass (A), and the numbers
of PCNA1 (B) and TUNEL1 (C) ASM cells versus basement membrane length. Control horses: circles and flat (thin) linear regression functions. Heaves: triangles and thick regression curves. In
heaves, the increments in ASM mass (A) and proliferating cells
(B) are larger the smaller the airways.
difference in basement membrane length) in each study
group separately. This approach is limited by the fact
that PCNA immunostaining and TUNEL were performed
on different preparations, and only some airway sections
(n 5 36) met the inclusion criteria for this analysis, resulting in a low statistical power (53%). Thus, the weakness
386 Herszberg et al
J ALLERGY CLIN IMMUNOL
AUGUST 2006
that the increase in the frequency of apoptotic myocytes
may develop in the course of remodeling as a compensatory mechanism to limit the increase in ASM mass.
DISCUSSION
Mechanisms of asthma and
allergic inflammation
FIG 5. Regression analysis of (A) ASM mass and (B) apoptotic cells
versus proliferating ASM cells/mm2. *Outlier. C, Summary graph
of the increases in proliferating (7-fold) and apoptotic (6-fold)
myocytes and ASM mass (close to 3-fold). *P < .05 vs control.
of the association detected does not rule out the possibility
of a stronger relationship between smooth muscle cell
proliferation and apoptosis. Overall, the data reflect a
concomitant increase in the numbers of proliferating and
apoptotic ASM cells in heaves (Fig 5, C), suggesting
Human asthma involves a series of structural changes of
the airways that occur in association with chronic airway
inflammation and may result from sustained and abnormal
repair responses. Such changes, termed airway remodeling, have recently received considerable attention because
they likely underlie the pathogenic mechanisms leading to
airway hyperresponsiveness, abnormal airway narrowing,
and, eventually, irreversibly impaired lung function.14,15
Airway remodeling may occur from the early stages of
the disease, perhaps antedating symptoms. The extent of
the structural alterations may be closely related to the disease severity and largely account for the cases of difficult
asthma. Airway remodeling involves alterations such as
goblet cell and mucous gland hyperplasia, subepithelial
fibrosis, neovascularization, ASM growth, and an overall
thickening of the airway wall.1 The increase in ASM mass
is a particularly significant change that has been observed
in human beings with asthma16-20 and experimentally
reproduced in rodent models of allergic sensitization
and repeated airway challenge.5,21-25 In the BN rat, the
in vivo modeling of ASM growth suggests that myocyte
hyperplasia contributes at least in part to the increase in
ASM mass.6 Mathematical modeling of airway mechanics,2-4 and experimental data correlating the ASM mass
with the responsiveness to challenge with cholinergic agonists,5 suggest that the growth of ASM is sufficient to
explain airway hyperresponsiveness. Although the ASM
may be intrinsically normal in asthma, an increased ASM
mass may increase airway narrowing through an augmentation in force generation and by an amplification of
the effect of smooth muscle fiber shortening.2-4
Despite the ability to reproduce ASM remodeling in
experimental asthma, the models used, based almost exclusively on rodents, would benefit from further assessment of the extent to which their features can be translated
to human asthma. Two issues need to be considered in this
regard. One is the possible influence of animal size on the
dynamics of cell turnover and its potential relevance to the
roles of proliferation and apoptosis. The other is the ability
of rodent models, where induced sensitization is followed
by repeated airway challenge within a relatively short
period, to represent the chronicity of naturally occurring
disease, and the long-term alterations associated with it. In
this regard, heaves is an spontaneously occurring disease
that affects a large mammal and closely parallels human
asthma.7,26 Tools such as bronchial biopsy, percutaneous
lung biopsy, or a thoracoscopically guided wedge pulmonary resection are feasible and can be used safely in
horses.27,28 Moreover, the prospective study of horse
cohorts with controlled exposure to moldy hay in an
experimental setting may allow the analysis of disease
mechanisms in early asymptomatic stages, an approach
not feasible in human beings. The airways of horses with
heaves show many of the pathophysiological features of
extrinsic human asthma. There is chronic airway inflammation, episodes of airway obstruction induced by antigen
inhalation (moldy hay), and airway hyperresponsiveness
to histamine and methacholine.7 Similar to asthma, the
symptoms are relieved by b2-adrenergic agonists and
corticosteroids.29 The airway inflammatory infiltrates are
associated with a TH2 activation phenotype,30 and the affected horses have a positive passive cutaneous anaphylaxis test31 and elevated levels of IgE in bronchoalveolar
lavage10,11 and serum.12 A difference with human asthma
is that the infiltrating granulocytes are predominantly neutrophils,7,30 although eosinophils are also present in the
airway tissues. Heaves is triggered by moldy hay, but it
is not a form of extrinsic allergic alveolitis.
In the current study, our data show for the first time
that heaves also parallels human asthma in terms of the
presence of airway remodeling involving the smooth
muscle, and suggest that similar mechanisms of ASM
growth may be shared. Compared with disease-free horses,
the horses with heaves had a significant increase in ASM
mass associated with evidence of increased myocyte
proliferation. Furthermore, a correlation between the ASM
mass and the frequency of PCNA1 ASM cells was found.
These data suggest that myocyte hyperplasia may contribute
to the ASM growth in heaves. In human asthma, the growth
of ASM has been attributed to both hyperplasia and cell hypertrophy,17,32,33 although some uncertainty remains regarding the ability to detect cell proliferation markers (proteins
expressed during the S phase of the cell cycle) by immunohistochemistry in bronchial biopsies of patients with
asthma.34 Compared with the analysis of whole airway
wall sections, the detection of proliferation markers in superficial bronchial biopsies may be less than ideal to capture the
positive ASM cells, which may be relatively low-frequency
events. ASM cells obtained from bronchial biopsies of patients with asthma, however, showed higher proliferation
rates when expanded in culture,35 consistent with earlier reports of an association between airway hyperresponsiveness
and increased sensitivity of ASM to growth factors.36
Our data show an increase in the rate of apoptotic ASM
cells in heaves. The role of apoptosis in the physiological
turnover of smooth muscle cells has not been defined to
date. Some studies have suggested that receptor-mediated
apoptosis may play a role in the control of myocyte
numbers because both airway and vascular smooth muscle
cells constitutively express Fas and undergo apoptosis on
Fas ligation in vitro.37,38 In atherosclerosis, the progression of atheromatous plaques involves T-cell inflammatory infiltrates in the arterial wall and growth of the
vascular smooth muscle associated with an increase in myocyte apoptosis.37 Our data showing an increase in ASM
mass associated with increases in both myocyte proliferation and apoptosis in heaves suggest a role for increased
myocyte apoptosis as a compensatory mechanism for the
hyperplastic growth of ASM.
The influence of airway size in remodeling is unclear,
and data from separate studies differ. Here we observed
an increase in ASM mass and PCNA1 and TUNEL1
myocytes at all airway sizes, although the increments in
ASM mass and PCNA1 cells were progressively greater
the smaller the airways. In the BN rat, the growth of ASM
was greater in larger airways in actively sensitized animals,5 whereas an adoptive transfer T-cell–driven model
showed an influence of airway size similar to the pattern
detected here in horses.39 In human asthma, the pattern
of anatomic distribution of ASM growth, and the relative
contribution of myocyte hyperplasia and hypertrophy to
ASM accumulation at different airway sizes, remain controversial.33 The variety of patterns seen in different studies and models may result from varying observational
conditions and techniques, and may also reflect varying
factors such as the sites of preferential deposition of allergen, the function of antigen presenting cells, and the local
inflammatory activity.
In summary, heaves, an asthma-like equine disease,
involves airway remodeling. As in human beings with
asthma and rodent models of experimental asthma, there
is an increase in the ASM mass that may underlie the
mechanism of airway hyperresponsiveness and airflow
obstruction in the affected horses. Myocyte hyperplasia
may cause at least in part the abnormal growth of ASM,
and a concomitant increase in apoptosis may play a
compensatory role. Because heaves is a naturally occurring condition, it may be a suitable model of spontaneous
disease onset, chronicity, and ASM remodeling in a large
mammal. This model creates an opportunity for studies
aimed at filling current gaps in our knowledge such as the
time course of remodeling during early disease, when
the initiation of structural changes in the airways may
antedate symptoms or detectable changes in lung function.
The conclusions from such studies may be useful in the
testing of early therapeutic interventions of relevance to
human asthma.
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